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Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3), which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2) in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes.

Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.

The integrin alpha6beta1 and its main ligand laminin-111 are overexpressed in glioblastoma, as compared with normal brain tissue, suggesting they may be involved in glioblastoma malignancy. To address this question, we stably expressed the alpha6 integrin subunit in the U87 cell line via retroviral-mediated gene transfer. We show that cell surface expression of the alpha6beta1 integrin led to dramatic changes in tumor U87 cell behavior, both in vitro and in vivo. Nude mice receiving either subcutaneous or intracerebral inoculation of alpha6beta1-expressing cells developed substantially more voluminous tumors than mice injected with control cells. The difference in tumor growth was associated with a marked increase in vascularization in response to alpha6beta1 integrin expression and may also be related to changes in the balance between cell proliferation and survival. Indeed, expression of alpha6beta1 enhanced proliferation and decreased apoptosis of U87 cells both in the tumor and in vitro. Additionally, we demonstrate that alpha6beta1 is implicated in glioblastoma cell migration and invasion and that laminin-111 might mediate dissemination of alpha6beta1-positive cells in vivo. Our results highlight for the first time the considerable role of the integrin alpha6beta1 in glioma progression.

Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins alphavbeta5/beta6 and alpha2beta1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our understanding of the consequences of alphavbeta5/beta6 function on alpha2beta1, we decreased the expression of alphav integrins by either siRNA or lysosomal targeting strategies or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced alpha2beta1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The alphavbeta5/beta6-dependent limitation of alpha2beta1 function could be overridden by TS2/16, an activating anti-beta1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high alpha2beta1-mediated cell migration observed in the absence of alphav integrins or following activation of alpha2beta1 integrins by the TS2/16. These results suggest that integrins alphavbeta5/beta6 repress alpha2beta1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of alpha2beta1-mediated migration.

Integrin engagement regulates cell adhesion, shape, migration, growth, and differentiation, but molecular mechanisms coordinating these functions in cells remain unclear. Because of their migratory and differentiation potential, neural crest cells constitute a powerful paradigm to address this question. Here, we describe that laminin-1, a major component of their migration routes, promotes crest cell spreading, migration and survival through two distinct integrin-binding domains that are situated on both sides of its alpha1 subunit and can be separated in the LN-1 elastase proteolytic fragments E1' and E8. Interaction with either domain was mediated by the same integrin alpha1beta1 but produced distinct, complementary responses through specific signaling cascades. FAK activation upon E8 binding induced spreading, formation of actin bundles and focal adhesions, stimulated oriented migration, but failed to support survival. Conversely, Erk activation upon E1' binding promoted long-term survival and random migration without actin reorganization. Consistent with this, interaction with laminin-5 or laminin-10/11, which do not harbor integrin-binding domains in the N-terminal side of their alpha chains, failed to support survival. Thus, the signaling activity and function of integrins might depend on binding domains in their ligands, thereby revealing ligand control of integrin function as a possible mechanism for the modulation and coordination of cell response to adhesive signals.

We present a general strategy for the dominant negative reduction in the levels of type-1 membrane-bound heterodimeric proteins within the secretory pathway through fusion of the soluble ectodomain of one of the partners to the transmembrane-cytosolic tail of the lysosomal protein Lamp1. Thus, in human embryonic kidney (HEK)-293 cells, overexpression of an integrin beta 3Lamp1 chimera resulted in a drastic reduction of its endogenous partner, the integrin alpha v subunit. The mechanism involves the formation in the endoplasmic reticulum of a alpha v/beta 3Lamp1 complex that is subsequently sorted towards a lysosomal/endosomal degradation pathway. The specificity of this approach is afforded by the invariance in the levels of the endogenous integrins alpha 5 and beta1 as compared with control cells. Conversely overexpression of integrin beta 3 in HEK-293 cells led to an increased level of alpha v beta 3 at the cell surface. Functionally beta 3Lamp1 and beta 3 overexpressors exhibit decreased and increased adhesion to vitronectin, respectively, as well as diminished cellular aggregation. The application of this technology should enable the analysis of the functional importance of homodimers or heterodimers in the cell types of choice and the identification of novel partner proteins by proteomic approaches.